Lower Ct for gene of interest than housekeeping gene - (Jul/14/2005 )
hello all
we have a problem concidering a real time pcr curve:
it seems that the gene we are trying to determine its expression has a lower Ct than the housekeeping gene (18s). (higher expression than the housekeeping gene)
will it be possible, under these circumstances to continue using the deltadelta Ct method?
thanks
we have a problem concidering a real time pcr curve:
it seems that the gene we are trying to determine its expression has a lower Ct than the housekeeping gene (18s). (higher expression than the housekeeping gene)
will it be possible, under these circumstances to continue using the deltadelta Ct method?
thanks
I would like to know the answer to this qn too....help!!!!
Absolutly without question. In my published real time results, most all of my genes of interest have a lower Ct value than the house keeping gene.
Housekeeping genes almost by definition are abundant.. thats why we use them. So long as there is less then 2 cycle difference between the samples you are using to correct to the housekeeping gene, you are just fine.
An added caveat.. 18S is very abundant.. that is why I don't use it. It passes threshold at around cycle 8-10.... which is very early and makes it harder to use as a corrector... unless you dilute your cDNA. I once had to use 18S as GAPDH message was affected by treatment and diluted the cDNA 1:1000 and used it against 1:10 diluted cDNA for my gene of interest.
The important thing is to get your message to pass threshold between cycle 15-25 as a rule of thumb.
Housekeeping genes almost by definition are abundant.. thats why we use them. So long as there is less then 2 cycle difference between the samples you are using to correct to the housekeeping gene, you are just fine.
An added caveat.. 18S is very abundant.. that is why I don't use it. It passes threshold at around cycle 8-10.... which is very early and makes it harder to use as a corrector... unless you dilute your cDNA. I once had to use 18S as GAPDH message was affected by treatment and diluted the cDNA 1:1000 and used it against 1:10 diluted cDNA for my gene of interest.
The important thing is to get your message to pass threshold between cycle 15-25 as a rule of thumb.
Yes, I've used 18s and it did come up very early, but I got around the problem by reducing 18s primer concentration. I usually use 50nm for the reaction, but for 18s I used 25nm, which gave me Ct values ard 15. But reducing the primer concentration further did not help...only gave me results all over the place.
Do not reduce primer concentration. I cannot stress this enough. Primer is not supposed to be limiting in this rxn. Only cDNA. Having a primer concentration from your control different from your gene of interest invalidates the results. The only thing that should be limiting in the reaction during log amplification is cDNA.
Only reduce your cDNA mass.
Do not reduce primer concentration. I cannot stress this enough. Primer is not supposed to be limiting in this rxn. Only cDNA. Having a primer concentration from your control different from your gene of interest invalidates the results. The only thing that should be limiting in the reaction during log amplification is cDNA.
Only reduce your cDNA mass.
.
Thanks pBluescript. That's an enlightening point. How about increasing primer conc for genes that come up late? i.e. instead of 36, i want them to come up at 25-28, so can i increase primer conc from 50nm to 100nm? Thanks in advance!
Do not reduce primer concentration. I cannot stress this enough. Primer is not supposed to be limiting in this rxn. Only cDNA. Having a primer concentration from your control different from your gene of interest invalidates the results. The only thing that should be limiting in the reaction during log amplification is cDNA.
Only reduce your cDNA mass.
I read somewhere that says we should reduce or limit primer concentration of the abundant genes in a multiplex reaction. Does this only matter for multiplex reactions and not for single reactions?
I have not done this, but I seriously doubt it will help. Primer is already in great abundace compared to dna of interest. If you want you Ct values to come up earlier, do not dilute your cDNA or use more RNA in your rt rxn.
I have not done multiplex rtpcr, so I can't speak to that.
pBluescript or anyone else -
I was just wondering if you had published any papers or knew of any that you could refer me to where this technique of diluting cDNA for HKG's had been used.
Thanks
Meg
i've been told a million times in the last week we should be diluting cDNA for HKG and GOI as well [1 in 10 if you use a 20ul RT volume]